Sugar-producing and texture-improving bakery methods and products formed therefrom

ABSTRACT

Novel yeast-raised and other bakery products and methods of making those products are provided. The products are formed from dough having a thermally-stable amyloglucosidase, and a raw starch degrading amyloglucosidase and/or an anti-staling amylase. The level of added sugar included in the dough can be substantially reduced, and even eliminated, while still achieving a sweet product. Additionally, the resulting bakery product is free of, or at least substantially free of, fructose. The final baked product will also have improved texture properties, including superior firmness, resilience, and adhesiveness and can be made with a reduced amount of yeast.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention is broadly concerned with the preparation ofbakery products by incorporating a specific enzyme formulation thatgenerates sugar during baking Advantageously, the final product is freeof, or substantially free of, added sugar and fructose, while stillhaving a taste and flavor equal to or better than equivalent productsmade with added sugar. Additionally, the present invention significantlyimproves the textural quality and shelf-life of the bakery productsthrough the synergistic interactions of the included enzymes.

Description of the Prior Art

Bakery products are generally appealing to consumers due to theirfreshness and sweet taste. With prior art products, this is due to theaddition of sugars, such as sucrose, high fructose corn syrup, honey,etc., to the ingredients used to form the products. Recently, addedsugar has been singled out as one of the unhealthiest ingredients infood. Added sugars contain high levels of fructose (generally 50%),which has been associated with potential health risks. Fructose ismetabolized in the liver, resulting in harmful end products liketriglycerides, uric acid, and free radicals. This can lead to healthailments such as non-alcoholic fatty liver disease, increased LDLcholesterol, cardiovascular disease, gout, and/or higher triglycerides,among other things.

Thus, it would be preferable to avoid adding added sugars, especiallyfructose-containing added sugars, to these products. However, the sweettaste is desirable for the products to be appealing, so simply notadding sugar would lead to products having a poor taste and lackingconsumer appeal. The use of artificial sweetening products in place ofsugar leads to problems of its own, including potential health issues, ataste that is not appealing to some people, and the consumer-unfriendlyingredient labeling. Also, while sugar alcohols have become a popularway to sweeten products, they also do not have as appealing of a tasteas typical sugars, and many people cannot digest sugar alcoholsproperly.

There is a need for a process for forming these products that does notrequire the inclusion of the added sugars. Furthermore, it would behighly desirable if the final products were free of, or substantiallyfree of, fructose while still having an appealing and sweet taste.

SUMMARY OF THE INVENTION

The present invention is broadly concerned with a method of forming abakery product, where the method comprises providing a dough comprising:

yeast;

an initial quantity of sugar;

a source of starch; a thermally-stable amyloglucosidase that exhibitsactivity at temperatures at which the starch gelatinizes; and

an enzyme selected from the group consisting of:

raw starch degrading amyloglucosidases;

anti-staling amylases; and

mixtures thereof.

The dough is baked for a time and temperature sufficient to yield thebakery product, with the bakery product having a final quantity of sugarthat is greater than the initial quantity of sugar.

The invention also provides a dough useful for forming a yeast-raisedbakery product and comprising a source of starch, yeast, and water. Theimprovement is that the dough comprises a thermally-stableamyloglucosidase that exhibits activity at temperatures at which thestarch gelatinizes, and an enzyme selected from the group consisting of:

raw starch degrading amyloglucosidases;

anti-staling amylases; and

mixtures thereof.

In a further embodiment, the invention provides a yeast-raised bakeryproduct formed from flour, yeast, and water. The improvement is that theproduct comprises:

-   -   an inactivated, thermally-stable amyloglucosidase derived from a        thermally-stable amyloglucosidase that exhibits activity at        temperatures at which starch gelatinizes;    -   at least about 5% by weight sugar, based upon the total weight        of the bakery product taken as 100% by weight; and    -   less than about 0.5% by weight fructose, based upon the total        weight of the bakery product taken as 100% by weight.    -   A further improvement is that the bakery product comprises a        decrease in crumb firmness by at least about 50%, preferably at        least about 75%, and more preferably from about 90% to about        100%, when compared to the same product formed from ingredients        where 8% added sugar is included in the initial ingredients and        either without the enzyme formulations of the present invention        or with a current market standard anti-staling enzymatic        product, such as the Ultra Fresh Premium 250 from Corbion        illustrated in FIG. 7 and FIG. 11.    -   The bakery product furthermore comprises an improvement of crumb        resilience by at least 10%, preferably at least about 15%, and        more preferably from about 20% to about 28%, when compared to        the same product formed from ingredients where 8% added sugar is        included in the initial ingredients and with a conventional        enzyme, such as AMG 1100 from Novozymes, or a current market        standard anti-staling enzymatic product, such as the Ultra Fresh        Premium 250 from Corbion illustrated in FIG. 2 and FIG. 12.    -   Yet a further improvement of the bakery product is a decrease of        crumb adhesiveness by at least 10%, preferably at least about        25%, and more preferably from about 25% to about 50%, when        compared to the same product formed from ingredients where a        conventional enzyme, such as AMG 1100 from Novozymes, was        included, as illustrated in FIG. 3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph comparing the sugar production capabilities of aconventional RSD amyloglucosidase (AMG 1100) to a thermally-stableamyloglucosidase (Po-AMG) from Example 1;

FIG. 2 is a graph comparing the bread resilience modificationcapabilities of a conventional RSD amyloglucosidase (AMG 1100) to athermally-stable amyloglucosidase (Po-AMG) from Example 1;

FIG. 3 is a graph comparing the bread adhesiveness modificationcapabilities of a conventional RSD amyloglucosidase (AMG 1100) to athermally-stable amyloglucosidase (Po-AMG) from Example 1;

FIG. 4 is a graph illustrating that both a conventional RSDamyloglucosidase (AMG 1100) and a thermally-stable amyloglucosidase(Po-AMG) can be used to produce small amounts of sugar during the doughmixing and dough proofing stages from Example 2;

FIG. 5 is a graph illustrating the total amounts of glucose produced byeither a RSD amyloglucosidase (AMG 1100), or a thermally-stableamyloglucosidase (Po-AMG), or the combination of the two in finishedbread from Example 2;

FIG. 6 is a graph illustrating that a significant amount of glucose canonly be produced by a thermally-stable amyloglucosidase (Po-AMG),whereas the conventional amyloglucosidase (AMG 1100) was not able toproduce a significant amount of glucose during baking from Example 2;

FIG. 7 is a graph showing the effects of reducing the added sugar (inthis case sucrose) in dough formulas on the performance of anti-stalingenzymes in terms of reducing the crumb firmness from Example 3;

FIG. 8 is a graph showing the effects of reducing the added sugar (inthis case sucrose) in dough formulas on the performance of anti-stalingenzymes in terms of the amount of the enzyme end-product (i.e., maltose)produced in the bread from Example 3;

FIG. 9 is a graph comparing the glucose, fructose, and maltose contentsin various bread formulations in Example 4;

FIG. 10 is a graph of the relative sweetness of the different breadformulations in Example 4;

FIG. 11 is a graph of the firmness of the different bread formulationsin Example 4;

FIG. 12 is a graph of the resilience of the different bread formulationsin Example 4;

FIG. 13 shows sensory results comparing a control bread to the testbread formulated in Example 5;

FIG. 14 provides sensory evaluation results showing the sweetness of acontrol bread compared to the test bread formulated in Example 5;

FIG. 15 shows sensory preference results of a control bread compared tothe test bread formulated in Example 5; and

FIG. 16 is a graph showing the sugar contents of a control breadcompared to a test bread according to the invention in Example 5.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In more detail, the present invention is concerned with novel doughformulations as well as novel methods of making yeast-raised, bakeryproducts, and other bakery products with these formulations. Theseproducts include those selected from the group consisting of breads,pretzels, English muffins, buns, rolls, tortillas (both corn and flour),pizza dough, bagels, and crumpets.

In the inventive methods, ingredients for the particular product aremixed together. Typical ingredients and their preferred ranges are setforth in Table 1.

TABLE 1 MOST INGREDIENT BROAD RANGE* PREFERRED* PREFERRED* YeastCompressed from about 1% to about from about 2% to about from about 3%to 10% 6% about 4% Dough Strengthener from about 0% to about from about0.25% to from about 0.35% to 2% about 1% about 0.5% Added Sugar** lessthan about 10% less than about 3% about 1% Dry Milk from about 0% toabout from about 1% to about from about 1% to 3% 2% about 1.5% Salt(typically from about 1% to about from about 1.5% to from about 1.75% toNaCl) 3% about 2.5% about 2.25% Mold Inhibitor from about 0% to aboutfrom about 0.1% to from about 0.25% to 1% about 0.5% about 0.35% Oil/Fatfrom about 0% to about from about 1% to about from about 2% to 20% 6%about 3% Flour Improver from about 0 ppm to from about 10 ppm to fromabout 40 ppm to about 500 ppm about 200 ppm about 75 ppm Emulsifiersfrom about 0% to about from about 0.5% to from about 1% to 4% about 3%about 2.5% Water from about 50% to from about 55% to from about 58% toabout 75% about 70% about 65% Thermally-Stable at least about 300 fromabout 500 to from about 750 to Amyloglucosidase AGU/kg flour about 1,500AGU/kg about 1,250 AGU/kg flour flour Raw Starch from about 0 to aboutfrom about 100 to from about 500 to Degrading 5,000 AGU/kg flour about2,500 AGU/kg about 1,000 AGU/kg Amyloglucosidase flour flour BacterialAmylase from about 0 to 20,000 from about 1,000 to from about 3,000 toMANU/kg flour about 10,000 about 5,000 MANU/kg flour MANU/kg flour OtherEnzymes from about 0 ppm to from about 20 ppm to from about 100 ppmabout 2,000 ppm about 300 ppm to about 200 ppm *Percentage or ppm basedupon the weight of flour. **Refers to all types of added sugar presentin the formulation. Sugars that can be added to the formulation includesucrose, glucose, fructose, high fructose corn syrup, honey, brownsugar, lactose, galactose, maple syrup, and rice syrup. “Added sugar”does not include sugar that could be inherently present in otheringredients (e.g., as part of the flour) in the dough mixture, nor doesit include sugar alcohols (e.g., xylitol, sorbitol) or artificialsweetening ingredients.

In a particularly preferred embodiment, the added sugar is about 0% byweight, and in another embodiment the added sugar is 0% by weight.

MANUs and AGUs are measures of the enzymatic activity of an amylase andan amyloglucosidase, respectively. As used herein, one unit of MANU(Maltogenic Amylase Novo Unit) is defined as the amount of enzymerequired to release one μmol of maltose per minute at a concentration of10 mg of maltotriose (Sigma M 8378) substrate per ml of 0.1 M citratebuffer, pH 5.0 at 37° C. for 30 minutes. One unit of AGU(Amyloglucosidase Unit) is defined as the amount of enzyme required tohydrolyze 1 μmol maltose per minute at a substrate concentration of 100milimole maltose in a 0.1 M acetate buffer, pH 4.3 at 37° C. In eitherinstance, the amounts of maltose in μmols can be determined by comparingthe final solution to a standard maltose solution.

In addition to the ingredients from Table 1, the dough will include asource of starch, such as those selected from the group consisting ofwheat flour, rye flour, oat flour, barley flour, triticale flour, riceflour, tapioca starch, corn starch, wheat starch, rice starch, potatostarch, corn flour, and potato flour. The source of starch willtypically be included to provide levels of from about 50% to about 95%by weight starch, and preferably from about 65% to about 85% by weightstarch, based upon the total weight of the flour taken as 100% byweight. When flour is the source of starch, this will typically resultin flour levels of from about 40% to about 70% by weight flour, andpreferably from about 50% to about 60% by weight flour, based upon thetotal weight of the dough taken as 100% by weight.

The yeast used can be any yeast conventionally used in yeast-raisedbakery products, with cream and compressed yeast being preferred.Suitable dough strengtheners include those selected from the groupconsisting of sodium stearoyl lactylate, ethoxylated monoglyceride,diacetyl tartaric acid esters of mono- and diglycerides (DATEM), andmixtures thereof.

Preferred mold inhibitors include those selected from the groupconsisting of calcium and/or sodium propionate, potassium sorbate,vinegar, raisin juice concentrate, and mixtures thereof. The preferredoil or fat is selected from the group consisting of soy oil, partiallyhydrogenated soy oil, lard, palm oil, corn oil, cottonseed oil, canolaoil, and mixtures thereof.

Suitable flour improvers include those selected from the groupconsisting of ascorbic acid, potassium bromate, potassium iodate,azodicarboamide, calcium peroxide, and mixtures thereof. While anyconventional emulsifier can be utilized, preferred emulsifiers includepolyoxyethylene sorbitan monostearate (typically referred to asPolysorbate 60) and monoglycerides, such as powdered and hydratedmonoglycerides, citrated monoglycerides, and succinylatedmonoglycerides.

Thermally-Stable Amyloglucosidases

The dough will also include a thermally-stable amyloglucosidases. Thethermally-stable amyloglucosidase utilized in the present inventionshould be selected so that it is active and available to act on starchas it gelatinizes during the baking process. That is, the bulk of thestarch present in the dough prior to baking is in the form of a starchgranule, which is not readily acted upon by enzymes. The raw starch willbegin to gelatinize at about 65° C. and is typically fully gelatinizedby around 85° C. Gelatinized starch is more easily hydrolyzed intoglucose by amyloglucosidases. Thus, the selected thermally-stableamyloglucosidase should be sufficiently heat-stable that it is able toact on the starch in the dough as the dough transitions to bread (i.e.,it should be active, or at least partially active, from about 65° C. toabout 85° C.). At the same time, it is preferred that the selectedthermally-stable amyloglucosidase is inactivated by the end of baking(i.e., about 95° C. to about 100° C.) as residual amyloglucosidaseactivity in fully baked products can negatively affect the quality ofthe final product during its shelf life.

Thus, thermally-stable amyloglucosidases for use in the presentinvention will have a half-life (T_(1/2)) of from about 1 minute toabout 30 minutes at about 85° C., preferably from about 3 minutes toabout 20 minutes at about 85° C., and more preferably from about 5minutes to about 15 minutes at about 85° C. These values are obtained ata pH of 4.5 and in 0.12 mM CaCl₂.

In one embodiment, the preferred thermally-stable amyloglucosidase willhave an optimum temperature of at least about 60° C., preferably fromabout 60° C. to about 85° C., more preferably from about 70° C. to about85° C., and even more preferably from about 75° C. to about 80° C., whenassayed at a pH of about 4.5. As used herein, “optimum temperature” ofan enzyme refers to the temperature at which the enzyme activity ishighest at the designated assay condition.

In one embodiment, the thermally-stable amyloglucosidases utilized willhave a residual enzyme activity of from about 25% to about 90%,preferably from about 35% to about 70%, and more preferably from about35% to about 60% after about 15 minutes incubation at 85° C. In order toavoid a negative impact on the cooked bread, the selectedthermally-stable amyloglucosidases will have a residual enzyme activityof less than about 15%, preferably less than about 10%, and morepreferably less than about 5% after about 3 minutes at 100° C. in a 5.0pH buffer with 0.12 mM CaCl₂. As used herein, “residual enzyme activity”is the enzymatic activity (in MANUs or AGUs, as defined above) remainingafter the particular enzyme has been subjected to the conditions setforth in this paragraph (i.e., “final activity). The “% residual enzymeactivity” is calculated by comparing the enzymatic activity (in MANUs orAGUs, as defined above) remaining after the particular enzyme has beensubjected to the conditions set forth in this paragraph (i.e., “finalenzymatic activity), to the enzymatic activity (again, in MANUs or AGUs)of the same enzyme prior to being subjected to these conditions (i.e.,“initial enzymatic activity). Thus,

${\% \mspace{14mu} {Residual}\mspace{14mu} {Activity}} = {\left( \frac{{Final}\mspace{14mu} {Enzymatic}\mspace{14mu} {Activity}}{{Initial}\mspace{14mu} {Enzymatic}\mspace{14mu} {Activity}} \right) \times 100.}$

In one embodiment, the thermally-stable amyloglucosidases utilized willhave an optimal pH (i.e., the pH at which the enzyme activity is highestat the designated assay condition) of from about 3.0 to about 7.0,preferably from about 4.0 to about 6.0, and more preferably from about4.5 to about 5.5 when assayed with 1 mM CaCl₂.

In one embodiment, the preferred thermally-stable amyloglucosidase willhave a pH stability range of from about 3.0 to about 7.0, preferablyfrom about 4.0 to about 6.0, and more preferably from about 4.5 to about5.5. pH stability is measured by first incubating the particular enzymeat the designated pH for 20 hours at 37° C. The retained enzyme activityis then assayed and compared to the original enzyme activity. Thepreferred thermally-stable amyloglucosidase will retain at least about70%, preferably at least about 90%, and more preferably from about 95%to 100% of its original activity in the pH stability ranges mentionedabove.

Specific examples of thermally-stable amyloglucosidases suitable for usein the present invention include amyloglucosidases derived from strains(i.e., encoded by a DNA sequence found in one of the strains) selectedfrom the group consisting of:

-   -   (a) Penicillium oxalicum (such as Po-AMG that described in        International Publication No. 2011/127802 by Novozymes,        incorporated by reference herein);    -   (b) Talaromyces emersonii (such as that described in        International Publication No. 2009/028448, incorporated by        reference herein);    -   (c) Talaromyces duponti (such as that described in U.S. Pat. No.        4,247,637, incorporated by reference herein);    -   (d) Talaromyces thermophilius (such as that described in U.S.        Pat. No. 4,587,215, incorporated by reference herein);    -   (e) Clostridium thermoamylolyticum (such as that described in EP        135,138, incorporated by reference herein); and    -   (f) Clostridium thermohydrosulfuricum (such as that described in        International Publication No. 1986/001,831, incorporated by        reference herein).

Although the above sets forth some preferred thermally-stableamyloglucosidases, any thermally-stable amyloglucosidase meeting theabove described properties can work with the present invention. Thisincludes amyloglucosidases from any natural source, as well as variantsmade through gene modification.

Raw Starch Degrading Amyloglucosidases

In a preferred embodiment, a raw starch degrading amyloglucosidase ispresent in the dough. A raw starch degrading amyloglucosidase acts onraw starch molecules. In one embodiment, this raw starch degradingamyloglucosidase preferably has a lower optimal temperature than thefirst amyloglucosidase described above. Also, this raw starch degradingamyloglucosidase only needs to be moderately thermally stable. That is,it may lose most of its activity when the dough temperature is above thestarch gelatinization temperature. In a preferred embodiment, sugar isgenerated by the raw starch degrading amyloglucosidase only in thedough, but not during baking That is, raw starch degrading enzymes (suchas those sold under the names AMG 300 and AMG 1100) lose most of theiractivity at temperature at which starch gelatinizes.

Preferred raw starch degrading amyloglucosidases will have heatstability up to about 70° C., but will preferably lose activity ratherrapidly above 70° C. Thus, preferred raw starch degradingamyloglucosidases for use in the present invention will have a half-life(T_(1/2)) of from about 1 minute to about 20 minutes at about 70° C.,preferably from about 3 minutes to about 15 minutes at about 70° C., andmore preferably from about 3 minutes to about 10 minutes at about 70° C.Preferably, the raw starch degrading amyloglucosidases utilized willhave a residual activity of at least about 5%, preferably at least about10%, and more preferably from about 10% to about 20% after about 15minutes at 70° C. In another embodiment, the raw starch degradingamyloglucosidase will have an optimum temperature of less than about 70°C., preferably less than about 65° C., more preferably from about 40° C.to about 65° C., more preferably from about 40° C. to about 60° C., andeven more preferably from about 45° C. to about 55° C., at a pH of about4.5.

Suitable raw starch degrading amyloglucosidases are disclosed inInternational Publication No. 2012/088303 and Purification andProperties of a Thermophilic Amyloglucosidase from Aspergillus niger, W.Fogarty et.al., Eur J Appl Microbiol Biotechnol (1983) 18:271-278,incorporated by reference herein. Those produced from Aspergillus arepreferred, and particularly preferred include those derived from strainsselected from the group consisting of Aspergillus niger (such as thatsold under the name AMG® 1100, by Novozymes, Denmark).

Anti-Staling Amylases

In another embodiment, a bacterial or anti-staling amylase is included.It is preferred that the amylase be one that is inactivated betweenabout 80° C. and about 90° C., because starch hydrolyzation by theanti-staling amylase occurs much more effectively when starch granulesget gelatinized during baking. The most preferred anti-staling amylaseis a maltogenic amylase, more preferably a maltogenic α-amylase, andeven more preferably a maltogenic a-exoamylase. The most preferred suchamylase is sold under the name NOVAMYL by Novozymes A/S and is describedin U.S. Pat. No. RE38,507, incorporated by reference herein. Thismaltogenic amylase is producible by Bacillus strain NCIB 11837, or oneencoded by a DNA sequence derived from Bacillus strain NCIB 11837 (themaltogenic amylase is disclosed in U.S. Pat. No. 4,598,048 and U.S. Pat.No 4,604,355, the contents of which are incorporated herein byreference). Another maltogenic amylase which may be used in the presentprocess is a maltogenic β-amylase, producible by Bacillus strain NCIB11608 (disclosed in EP 234 858, the contents of which are herebyincorporated by reference). Another suitable anti-staling enzyme for usein the present invention is available from DuPont Danisco under thenames POWERFresh® G4 and POWERFresh® G+. Additionally, U.S. PatentApplication Publication No. 2009/0297659 (incorporated by referenceherein) discloses suitable amylases.

Some of the other enzymes that can be included in the invention inaddition to the maltogenic amylase include those selected from the groupconsisting of fungal amylases, bacterial alpha-amylase from Bacillussubtilis, hemi-cellulases, xylanases, proteases, glucose oxidase, hexoseoxidase, lipase, phospholipase, asparaginase, and cellulases.

As noted above, in some embodiments, the invention utilizes only athermally-stable amyloglucosidase. In a preferred embodiment, theinvention utilizes a raw starch degrading amyloglucosidase or ananti-staling amylase in addition to the thermally-stableamyloglucosidase. In a particularly preferred embodiment, the inventionutilizes a thermally-stable amyloglucosidase, a raw starch degradingamyloglucosidase, and an anti-staling amylase. Of course, the embodimentcan be selected depending upon the user's preferences and the particularproduct to be prepared.

Method of Making Baked Products

In forming the dough according to the invention, the above ingredientscan be simply mixed together in one stage using the “no-time doughprocess,” or they can be subjected to the “sponge and dough process.” Inthe “no-time dough process,” all ingredients are added to a mixing bowlat the same time and mixed for a time period from about 5 to about 15minutes to form the mixed dough.

In the “sponge and dough” process, part of the flour (e.g., 55-75% byweight of the total flour) is mixed with water, yeast, and preferablythe dough strengthener (if utilized) and allowed to ferment for a timeperiod of from about 3 hours to about 4 hours. This forms the “sponge.”After this time period, the remaining ingredients are mixed with thesponge for a time period of from about 2 minutes to about 10 minutes toform the mixed dough. The mixed dough is preferably allowed to rest fora time period of from about 5 minutes to about 15 minutes before beingformed into the desired size pieces and placed in the baking pans. Thedough is then preferably allowed to proof at a temperature of from about40° C. to about 50° C. at a relative humidity of from about 65% to about95% for a time period of from about 50 minutes to about 70 minutes.

During proofing, the enzymes present will begin to act on the starch.Any raw starch degrading amyloglucosidase present will begin to act onthe raw starch, as will the thermally-stable amyloglucosidase,converting some starch into glucose.

Regardless of the embodiment, sugars (and particularly non-fructosesugars) are generated during the baking (and preferably also duringproofing) process by the enzyme blend utilized. That is, the startingingredients or dough will contain some “initial quantity” of sugar. Thatinitial quantity could be zero, such as in no added sugar formulations.Or, that initial quantity could be some low-sugar amount (e.g., 1-3%) oran amount as high as 10%, as described above. More specifically, theinitial quantity of sugar is about 10% by weight or less, preferablyless than about 3% by weight, more preferably less than about 1% byweight. In a particularly preferred embodiment, the initial quantity ofsugar is about 0% by weight, more particularly 0% by weight. Regardlessof the initial quantity, after baking the final product will have afinal quantity of total sugar that is greater than the initial quantity.For the purpose of the invention, sugar or sugars are understood toinclude sucrose, glucose, fructose, high fructose corn syrup, honey,brown sugar, lactose, galactose, maple syrup, and rice syrup, but notsugar alcohols or artificial sweetening ingredients.

In more detail, in some embodiments, the initial dough of the invention(i.e., prior to proofing) contains little to no sugar (beyond minoramounts of sugars found in any flour or starch by nature or beinginherently present due to the type of any flour or starch used), andparticularly little to no fructose (i.e., less than about 0.2% byweight, preferably less than about 0.1%, preferably about 0% by weight,and preferably 0% by weight of each, based upon the total weight of theinitial dough taken as 100% by weight). In one embodiment, the initialdough will also contain little to no glucose (in the same low quantitiesas set forth above for fructose in the initial dough). During proofing,both raw starch degrading amyloglucosidase and thermally-stableamyloglucosidase will convert certain amount of starch to glucose. Afterthe dough is proofed, there will typically be total sugar levels (i.e.,total glucose, fructose, and maltose) of at least about 1% by weight,preferably from about 1% to about 2% by weight, and more preferably fromabout 2% to about 3% by weight, based upon the total weight of theproofed dough taken as 100% by weight. The glucose levels in the proofeddough will typically be at least about 1% by weight, preferably fromabout 1% to about 2% by weight, and more preferably from about 2% toabout 3% by weight, based upon the total weight of the proofed doughtaken as 100% by weight. Thus, the glucose present in the dough afterproofing will generally increase from 0% (or close to 0%) to at leastabout 1%, preferably to about 1% to about 2%, and more preferably fromabout 2% to about 3% by weight, based upon the total weight of theproofed dough taken as 100% by weight. When there is at least someamount of glucose present in the initial ingredient mixture (i.e., theglucose present in the initial ingredients is greater than 0%, whilestill being within the limits set forth above), the total glucosepresent in the proofed dough will be at least about 5 times, preferablyat least about 10 times, and more preferably from about 10 to about 15times that of the glucose quantity present in the dough prior toproofing. Advantageously, the fructose levels noted above will remainsubstantially unchanged. That is, the proofed dough will still have lessthan about 0.2% by weight fructose, preferably less than about 0.1% byweight fructose, and more preferably about 0% by weight fructose, basedupon the total weight of the proofed dough taken as 100% by weight.

After proofing, the product can then be baked using the times andtemperatures necessary for the type of product being made (e.g., fromabout 190° C. to about 220° C. for about 20 minutes to about 30minutes). While any non-thermally-stable enzymes, including any rawstarch degrading amyloglucosidases that were included in the originalingredients will still be present in their active forms during proofing,they will begin to be inactivated during baking, leaving behind theenzyme skeletons. However, the thermally-stable amyloglucosidase(s) andthe anti-staling amylase included in the initial ingredients will stillbe present in its active form as baking is commenced. Thus, as thestarch granules gelatinize during baking, the thermally-stableamyloglucosidase will be able to continue to hydrolyze the gelatinizedstarch, further producing glucose in higher quantities, whereas theanti-staling amylase will also continue to hydrolyze the gelatinizedstarch, leaving an anti-staling effect in the finished product, and alsoproducing maltose, other oligosaccharides, and dextrins. However, by theend of the bake cycle, both the thermally-stable amyloglucosidase andthe anti-staling amylase will be inactivated.

The invention results in a number of advantages, in addition to thosediscussed previously. The present invention results in the use ofsignificantly less yeast than in prior art products. Thus, using thepreviously mentioned enzyme formulations of the present invention yieldsa yeast reduction of at least about 15%, preferably at least about 20%,and more preferably from about 20% to about 35%, when compared to thesame product formed from ingredients where sugar is added to the initialingredients and without the enzyme formulations of the presentinvention. For example, when a dough with 0% added sugar in the initialingredients is utilized in combination with the enzyme formulations ofthe present invention, the above yeast reductions are achieved whencompared to the same product formed from ingredients where 8% addedsugar is included in the initial ingredients and without the enzymeformulations of the present invention.

An additional advantage of the present invention is the increasedfunctionality of the anti-staling maltogenic amylase (measured as crumbfirmness). That is, the use of a thermally-stable amyloglucosidaseallows for lower quantities of sugar, such as sucrose, to be added tothe starting dough, which in turn improves the performance ofanti-staling amylases, since most of the added sugars inhibit theanti-staling maltogenic amylase. It was observed that a dough with theenzyme formulations of the present invention and 0% added sugar in theinitial ingredients yields a decrease in crumb firmness by at leastabout 50%, preferably at least about 75%, and more preferably from about90% to about 100%, when compared to the same product formed fromingredients where 8% added sugar is included in the initial ingredientsand either without the enzyme formulations of the present invention, orwith a current market standard anti-staling enzymatic product, such asthe Ultra Fresh Premium 250 from Corbion, both of which are illustratedin FIG. 7 and FIG. 11.

Additionally, even though little to no added sugars (and, therefore,little to no fructose) were included when forming the initial dough, thefinal baked product formed utilizing the enzyme formulations of thepresent invention is as sweet or sweeter, when compared to a 8% addedsugar (e.g. sucrose) control product in a sensory test. That is, thebaked product will typically have total sugar levels (mainly thenon-fructose-containing glucose and maltose) of at least about 5% byweight, preferably from about 6% to about 12% by weight, and morepreferably from about 8% to about 10% by weight, based upon the totalweight of the final, baked, bakery product taken as 100% by weight. Whenthere is at least some sugar present in the initial ingredients (i.e.,the amount of sugar in the initial ingredients is greater than 0%), thetotal sugars present in the final baked product will generally be atleast about 5 times, preferably at least about 10 times, and morepreferably from about 16 to about 20 times that of the total sugarspresent in the initial ingredient mixture.

The glucose levels in the final baked product will typically be at leastabout 3% by weight, preferably from about 3% to about 10% by weight, andmore preferably from about 4% to about 6% by weight, based upon thetotal weight of the bakery product taken as 100% by weight. Thus, whenthere is at least some amount of glucose present in the initialingredients, the glucose present in the dough in the final baked productwill generally be at least about 15 times, preferably at least about 20times, and more preferably from about 20 to about 30 times that of theglucose quantity present in the initial ingredient formulation.

Again, and advantageously, the fructose levels noted above will remainsubstantially unchanged. That is, the final baked product will have lessthan about 1% by weight fructose, preferably less than about 0.5% byweight fructose, and more preferably less than about 0% by weightfructose, based upon the total weight of the bakery product taken as100% by weight. It will be appreciated that this presents a significantadvantage over the prior art because the health risks associated withfructose consumption are avoided.

As discussed above, in one embodiment the invention involves the use ofa thermally-stable amyloglucosidase together with an anti-staling(maltogenic) amylase. Since both thermally-stable amyloglucosidases andanti-staling amylases have similar thermal stabilities and both remainactive after starch granules gelatinize, they work synergisticallyduring baking Advantageously, the presence of the thermally-stableamyloglucosidases not only increases the sweet taste of the bakedproducts, but also decreases the crumb adhesiveness and increases thecrumb resilience. The invention further allows for the level ofexpensive anti-staling amylases to be reduced, while improving thetexture and still achieving a sweet bread. Thus, bakery products formedaccording to the present invention not only have improved crumb texturedue to reduced firmness, reduced adhesiveness, and increased crumbresilience, but these products also have improved taste and flavor dueto the small sugars, such as glucose and maltose, produced by thethermally-stable amyloglucosidases and the anti-staling amylases.

Regardless of the embodiment, when subjected to the firmness (i.e.,crumb compressibility) test described in the TEST METHODS section below,bakery products according to the invention will give results of lessthan about 250 g of force at day 7, preferably less than about 200 g offorce, and even more preferably less than about 160 g of force.Furthermore, when subjected to the adhesiveness test described in thatsame section, bakery products according to the invention will give avalue of from about 5 g*mm to about 25 g*mm, preferably from about 5g*mm to about 20 g*mm, and more preferably from about 10 g*mm to about20 g*mm when measured at shelf life day 7. The percent resilienceachieved will be at least about 28%, preferably from about 30% to about40%, and more preferably from about 32% to about 37% when measured shelflife day 7. Finally, when the final baked product is bread, the specificvolume is at least about 5.5 g/cc³, preferably at least about 6.0 g/cc³,and more preferably at least about 6.5 g/cc³, in a 454 g piece of bread.The volume is determined by VolScan laser volumeter manufactured byStable Micro Systems.

The invention pertains in particular to the subject-matter in thefollowing clauses, without being limited thereto or thereby. Theembodiments presented in the following clauses can be combined asindicated, and can also be combined with other matter in the presentdescription, and with the claims at the end of this document.

1. A method of forming a bakery product, said method comprising:

providing a dough comprising:

-   -   yeast;    -   an initial quantity of sugar;    -   a source of starch;    -   a thermally-stable amyloglucosidase that exhibits activity at        temperatures at which the starch gelatinizes; and    -   an enzyme selected from the group consisting of:    -   raw starch degrading amyloglucosidases;    -   anti-staling amylases; and    -   mixtures thereof; and

baking the dough for a time and temperature sufficient to yield thebakery product, said bakery product having a final quantity of sugarthat is greater than said initial quantity of sugar.

The method of clause 1, wherein:

-   said initial quantity of sugar is less than about 1.0% by weight,    based upon the total weight of the dough taken as 100% by weight;-   said final quantity of sugar is at least about 5.0% by weight, based    upon the total weight of the bakery product taken as 100% by weight;    and-   said bakery product comprises less than about 0.5% by weight    fructose, based upon the total weight of the bakery product taken as    100% by weight.

3. The method of claus 1 or 2, said thermally-stable amyloglucosidasebeing active at temperatures of from about 65° C. to about 85° C.

4. The method of any of clauses1-3, wherein said enzyme is a maltogenicamylase, and said bakery product has an adhesiveness of from about 5g*mm to about 25 g*mm when measured at shelf life day 7.

5. The method of any of clauses 1-4, wherein said source of starch isflour.

6. The method of any of clauses 1-5, wherein said thermally-stableamyloglucosidase has an optimum temperature of at least about 60° C.

7. The method of any of clauses 1-6, wherein said thermally-stableamyloglucosidase has a half-life (T_(1/2)) of from about 1 minute toabout 30 minutes at about 85° C.

8. The method of any of clauses 1-7, wherein said thermally-stableamyloglucosidase is derived from strains selected from the groupconsisting of Penicillium oxalicum, Talaromyces emersonii, Talaromycesduponti, Talaromyces thermophilius, Clostridium thermoamylolyticum, andClostridium thermohydrosulfuricum.

9. The method of any of clauses 1-8, wherein said enzyme is a raw starchdegrading amyloglucosidase having a half-life (T_(1/2)) of from about 1minute to about 20 minutes at about 70° C.

10. The method of any of clauses 1-9, wherein said raw starch degradingamyloglucosidase is produced from Aspergillus.

11. The method of any of clauses 1-10, wherein said initial quantity ofsugar is less than about 3% by weight, based upon the total weight ofthe dough taken as 100% by weight.

12. The method of any of clauses 10, wherein said initial quantity isgreater than 0%.

13. The method of any of clauses 1-12, wherein said final quantity ofsugar is at least about 10 times that of the initial quantity of sugar.

14. The method of any of clauses 1-13, wherein said final quantity ofsugar is at least about 5% by weight, based upon the weight of thebakery product being taken as 100% by weight.

15. The method of any of clauses 1-14, wherein said bakery productcomprises less than about 1% by weight fructose, based upon the weightof the bakery product being taken as 100% by weight.

16. The method of any of clauses 1-15, wherein said bakery productcomprises about 0% by weight fructose, based upon the weight of thebakery product being taken as 100% by weight.

17. The method of any of clauses 1-16, further comprising proofing saiddough prior to said baking.

18. The method of clause 17, wherein said dough has an initial quantityof glucose prior to said proofing and a final quantity of glucose aftersaid proofing, said final quantity being at least about 5 times that ofsaid initial quantity.

19. The method of clause 17 or 18, wherein said dough has greater than0% by weight glucose prior to said proofing, based upon the total weightof the dough prior to said proofing taken as 100% by weight.

20. The method of any of clauses 17-19, wherein said dough has a finalquantity of glucose after said proofing, said final quantity being atleast about 1.0% by weight, based upon the total weight of the doughafter said proofing taken as 100% by weight.

21. The method of any of clauses 1-20, wherein said bakery product isselected from the group consisting of bread, English muffins, pretzels,buns, rolls, tortillas, pizza dough, bagels, and crumpets.

22. The method of any of clauses 1-21, where said bakery product has afirmness of less than about 250 g of force at shelf life day 7.

23. The method of claim 1-22, wherein said bakery product has a percentresilience of at least about 28% when measured shelf life day 7.

24. A dough useful for forming a yeast-raised bakery product andcomprising a source of starch, yeast, and water, characterized in thatsaid dough comprises:

a thermally-stable amyloglucosidase that exhibits activity attemperatures at which the starch gelatinizes; and

an enzyme selected from the group consisting of:

-   -   raw starch degrading amyloglucosidases;    -   anti-staling amylases; and    -   mixtures thereof.

25. The dough of clause 24, said thermally-stable amyloglucosidase beingactive at temperatures of from about 65° C. to about 85° C.

26. The dough of any of clauses 24-25, wherein said source of starch isflour.

27. The dough of any of clauses 24-26, wherein said thermally-stableamyloglucosidase has an optimum temperature of at least about 60° C.

28. The dough of any of clauses 24-27, wherein said thermally-stableamyloglucosidase has a half-life (Ti12) of from about 1 minute to about30 minutes at about 85° C.

29. The dough of any of clauses 24-28, wherein said thermally-stableamyloglucosidase is derived from strains selected from the groupconsisting of Penicillium oxalicum, Talaromyces emersonii, Talaromycesduponti, Talaromyces thermophilius, Clostridium thermoamylolyticum, andClostridium thermohydrosulfuricum.

30. The dough of any of clauses 24-29, wherein said enzyme is a rawstarch degrading amyloglucosidase having a half-life (T_(1/2)) of fromabout 1 minute to about 20 minutes at about 70° C.

31. The dough of any of clauses 24-30, wherein said raw starch degradingamyloglucosidase is produced from Aspergillus.

32. The dough of any of clauses 24-31, wherein said dough is unproofeddough.

33. The dough of clause 32, wherein said unproofed dough comprises lessthan about 3% by weight sugar, based upon the total weight of theunproofed dough taken as 100% by weight.

34. The dough of clause 32 or 33, wherein said unproofed dough comprisesless than about 0.5% by weight sugar, based upon the total weight of theunproofed dough taken as 100% by weight.

35. The dough of any of clauses 24-31, wherein said dough is proofeddough.

36. The dough of clause 35, wherein said proofed dough comprises atleast about 1.0% by weight sugar, based upon the total weight of theproofed dough taken as 100% by weight.

37. The dough of clause 35 or 36, wherein said proofed dough comprisesless than about 0.2% by weight fructose, based upon the total weight ofthe proofed dough taken as 100% by weight.

38. The dough of any of clauses 35-37, wherein said proofed doughcomprises about 0% by weight fructose, based upon the total weight ofthe proofed dough taken as 100% by weight.

39. A yeast-raised bakery product formed from flour, yeast, and water,characterized in that said product comprises:

-   -   an inactivated, thermally-stable amyloglucosidase derived from a        thermally-stable amyloglucosidase that exhibits activity at        temperatures at which starch gelatinizes;    -   at least about 5% by weight sugar, based upon the total weight        of the bakery product taken as 100% by weight; and    -   less than about 0.5% by weight fructose, based upon the total        weight of the bakery product taken as 100% by weight.

40. The bakery product of clause 39, wherein said inactivatedthermally-stable amyloglucosidase is derived from a thermally-stableamyloglucosidase that is active at temperatures of from about 65° C. toabout 85° C.

41. The bakery product of clause 39 or 40, wherein said inactivatedthermally-stable amyloglucosidase is derived from a thermally-stableamyloglucosidase having an optimum temperature is from about 60° C. toabout 85° C.

42. The bakery product of any of clauses 39-41, wherein said inactivatedthermally-stable amyloglucosidase is derived from a thermally-stableamyloglucosidase having a half-life (T_(1/2)) of from about 1 minute toabout 30 minutes at about 85° C.

43. The bakery product of any of clauses 39-42, wherein said inactivatedthermally-stable amyloglucosidase is derived from a thermally-stableamyloglucosidase that is derived from strains selected from the groupconsisting of Penicillium oxalicum, Talaromyces emersonii, Talaromycesduponti, Talaromyces thermophilius, Clostridium thermoamylolyticum, andClostridium thermohydrosulfuricum.

44. The bakery product of any of clauses 39-43, wherein said sugar ispresent at a level of from about 6% to about 12% by weight, based uponthe total weight of the bakery product taken as 100% by weight.

45. The bakery product of any of clauses 39-44, wherein said bakeryproduct comprises about 0% by weight fructose, based upon the totalweight of the bakery product taken as 100% by weight.

46. The bakery product of any of clauses 39-45, wherein said bakeryproduct has a percent resilience of at least about 28% when measuredshelf life day 7.

47. The bakery product of any of clauses 39-46, where said bakeryproduct has a firmness of less than about 250 g of force at shelf lifeday 7.

48. The bakery product of any of clauses 39-47, wherein said bakeryproduct has an adhesiveness of from about 5 g*mm to about 25 g*mm whenmeasured at shelf life day 7.

EXAMPLES

The following examples set forth preferred methods in accordance withthe invention. It is to be understood, however, that these examples areprovided by way of illustration and nothing therein should be taken as alimitation upon the overall scope of the invention.

Test Methods Texture Analysis

The bread texture was measured at day 7 and day 14. After baking, thebread was cooled to an internal temperature of 100° F. (50 minutes),then weighed, measured for volume, and stored in atemperature-controlled room at 72° F.+/−2° F. until testing. At thattime, the loaves were sliced one loaf at a time with an Oliver 16 bladeslicer to a thickness of 25 mm+/−2 mm to produce 10 slices per one poundloaf. The center four slices were tested using Texture Profile Analysis(TPA) procedure. The measuring instrument was a Texture Analyzer fromStable Micro Systems (TA-XT2 Texture Analyzer—25 kg load cell with 1 gresolution). The software running this instrument was Texture ExpertExceed version 2.64. The settings for running the TPA on the TextureAnalyzer for bread are in the table below.

Texture Analyzer Settings Test Mode and Option TPA Pre-Test Speed(mm/sec) 2.00 Test Speed (mm/sec) 1.00 Post Test Speed (mm/sec) 1.00Target Mode Strain Strain 25% 2^(nd) Strain 40% Time (sec) 3 HoldingTime on 2^(nd) Compression (sec) 3 Trigger Type Auto Trigger Force(grams) 5 Units Force Grams Units Distance Millimeters

It will be appreciated that one skilled in the art would be able toadjust these settings based upon the type of product being tested. Forexample, the Distance (depth of test in mm) could be adjusted dependingupon the type of product tested.

A TA-4 probe (1½ inch-38 mm diameter acrylic cylinder) was used, andgraph preferences were set to Time and auto range on the X axis, andForce and auto range on the Y axis.

The procedure for measuring the bread was to lay a single slice on theplatform of the Texture Analyzer, position it so the probe wasapproximately in the center of the slice and about 10 mm above thesurface, and start the test program. The test generated a graph that wasused to quantify adhesiveness, firmness, and resilience. Specifically,the adhesiveness, or adhesive value, is the negative area following theend of the second curve and representing the energy needed to withdrawthe probe from the slice. The firmness is the force point on the firstcurve corresponding to a punch depth of 25% of the slice thickness.Resilience is the ratio of the energy released from the slice when theprobe is lifted from the slice to the energy applied to the slice whenthe probe is compressing the slice (AACC Method 74-09).

Sugar Extraction and Analysis

The sugar content of both the dough and bread was tested by measuring a20 g sample of dough or bread crumb in a blending cup. Next, 80 g ofdistilled water was added, and the hand-held blender was used todisperse the dough or crumb completely. About 12 ml of the mixture waspoured into a 15 ml tube and placed on ice. The tube was thencentrifuged at 4,000 rpm for 10 minutes. The supernatant was thenremoved (making sure to obtain the clear solution in the middle of thetube) and then transferred into two microfuge tubes. For doughextraction, the supernatant was boiled in the microfuge tube for 1minute, and then cooled on ice. The microfuge tubes were centrifuged at12,000 rpm for 10 minutes. The resulting supernatant was thentransferred to two new labeled microfuge tubes, which were stored in therefrigerator until sugar analysis.

Sugar content in the samples was analyzed on Dionex Ultimate 3000 RSHPLC system with Dionex CarboPac PA1 column (4×250 mm) with a PA10 guardcolumn (4×50 mm). The electrochemical detector used was Dionex ED40 withThermo Scientific's disposable electrodes. The HPLC mobile phase A was50 mM NaOH, while the mobile phase B was 200 mM NaOH. All sugar sampleswere filtered through 0.4 μm filter before loading to the HPLC column.

Example 1 Amyloglucosidase Sugar Production and Texture Modification

A conventional raw starch degrading (“RSD”) amyloglucosidase, AMG 1100(from Novozymes®, North Carolina), was compared to a thermally-stableamyloglucosidase, Po-AMG (from Novozymes®), in bread baking for theirsugar production and crumb texture modification capabilities. A standardwhite pan bread formulation was prepared according to the followingprocess.

White Staight Pan Bread Ingredients % of flour weight Gram Flour 100.0700.00 DEPENDOX ® AXC^(A) 0.04 0.28 Salt 2.0 14.00 Granulated sucrose1.0 7.00 Calcium Propionate 0.3 2.10 Soy Oil 2.0 14.00 Yeast -compressed 7.00 49.00 Water 64.0 448.00 GMS 90 SS^(B) 1.0 7.00 SodiumStearoyl Lactylate 0.35 2.45 (SSL) UFP 250^(C) 0.50 3.50 Total Weight176.3 ^(A)A blend of ascorbic acid, azodicarbonamide (ADA), fungalenzymes, and wheat starch (available from Corbion, Lenexa, KS).^(B)Hydrated monoglycerides (emulsifier; available from Corbion).^(C)Ultra Fresh Premium 250 (shelf-life extending enzymes; availablefrom Corbion).

Amyloglucosidase Optimal Name AGU/g Temp Opt. pH Half Time AMG 11001100/g 65-70° C. 4-5 7 min @ 70° C. Po-AMG 1680/g 75-80° C. 4-5 120 min@ 70° C.; 10 min @ 85° C.The amounts of amyloglucosidase were varied accordingly, as shown in theTable below.

Formula Variations Dough # 2 3 4 5 6 7 8 1 AMG 1100 Po-AMG AGU/kg 01,000 2,000 5,000 500 1,000 2,000 5,000 flourThe ingredients were added to a Hobart mixer, and mixed on low for 1minute and then on high for 13 minutes. The mixer bowl was chilled bycirculating 20° C. chilling water through the cooling jackets of themixing bowl. After mixing, the dough was allowed to rest on a wood benchfor 10 minutes and then, divided, sheeted, and moulded according to theprocessing parameters in the table below.

Processing Parameters Targeted Dough Temperature 78° F. (circulating 20°C. chilling water) Floor Time 10 min Divided Dough Wt. 525 g Rest afterRounding 5 min Mould Type and Settings Mould Type Straight grain 2ndRoller 2   Pressure Bd. 2.8 Proofer Setting 104° F., 95% RH Proof TimeTime to a targeted height Baking Temp 420° F. Baking Time 20 min CoolingTime at Room Temperature 60 min

The moulded dough pieces were then placed into loaf pans and proofed tothe targeted height for around 55-60 min. Before baking, a sample ofeach proofed dough was taken for sugar extraction and analysis. Afterbaking, the loaves were left on a metal shelf for cooling for 60 minutesand then packed individually in plastic bags for shelf life analysis,which included textural analysis with a Texture analyzer and sugarcontent analysis as described above.

FIG. 1 compares the sugar production capabilities of a conventional RSDamyloglucosidase, AMG 1100, to a thermally-stable amyloglucosidase,Po-AMG. The results showed that the thermally-stable amyloglucosidase,Po-AMG, is more effective in producing glucose in the bread, mainly dueto its ability to continue converting starch into glucose after the rawstarch was gelatinized at temperatures above 65° C. FIGS. 2 and 3compare the bread texture modification capabilities of a conventionalRSD amyloglucosidase, AMG 1100, to a thermally-stable amyloglucosidase,Po-AMG. The results clearly show that the thermally-stableamyloglucosidase, Po-AMG, had much greater texture modificationcapability, in terms of increasing crumb resilience and reducing crumbadhesiveness.

Based on the data illustrated in FIG. 2, an improvement of crumbresilience by at least 10%, preferably at least about 15%, and morepreferably from about 20% to about 28%, can be achieved by includingvarious levels of Po-AMG in the dough, comparing to the same productformed from ingredients where a conventional enzyme, such as AMG 1100from Novozymes, was included in the dough. Meanwhile as illustrated inFIG. 3, a decrease of crumb adhesiveness by at least 10%, preferably atleast about 25%, and more preferably from about 25% to about 79%, can beachieved by including various levels of Po-AMG in the dough, comparingto the same product formed from ingredients where a conventional enzyme,such as AMG 1100 from Novozymes, was included in the dough.

Example 2 Analysis of Sugar Production Capability

A standard white pan bread formulation was prepared according to thefollowing formulation and the same processing parameters described inExample 1. All of the bread dough was made with 1% added-sugar andspecified amounts of amyloglucosidases.

White Staight Pan Bread Ingredients % of flour weight Gram Flour 100.0700.00 DEPENDOX ® AXC 0.04 0.28 Salt 2.0 14.00 Granulated sucrose 1.07.00 Calcium Propionate 0.3 2.10 Soy Oil 2.0 14.00 Yeast - compressed5.50 38.50 Water 64.0 448.00 GMS 90 SS 1.0 7.00 SSL 0.35 2.45 NOVAMYL ®3D^(A) 0.02 0.14 Total Weight 174.8 ^(A)An anti-staling enzyme fromNovozymes ®.

Formula Variations Dough # 1 2 3 4 AGU/kg flour AMG 1100 0 0 1000 1000Po-AMG 0 1000 0 1000

Again, samples of proofed dough were collected and flash frozen forsugar analysis. Dough sugar was extracted as described above, exceptthat 10 g of dough was dispersed in 90 g of water. The results are shownin FIGS. 4-6. Sugar content in the proofed dough and in the baked breadwas analyzed and compared to determine when the sugars were producedduring the process of bread making.

Sugar analysis 0 AGU/kg 1000 AGU/kg flour Po-AMG flour Po-AMG Glucose inproofed dough   0 AGU/kg flour AMG 1100 0.06% 1.23% 1000 AGU/kg flourAMG 1100 1.56% 2.60% Glucose in Bread   0 AGU/kg flour AMG 1100 0.61%4.44% 1000 AGU/kg flour AMG 1100 2.08% 5.81% Maltose in Dough   0 AGU/kgflour AMG 1100 1.92% 0.61% 1000 AGU/kg flour AMG 1100 0.00% 0.00%Maltose in Bread   0 AGU/kg flour AMG 1100 7.29% 5.60% 1000 AGU/kg flourAMG 1100 7.24% 5.76% Glucose Produced during Baking   0 AGU/kg flour AMG1100 0.55% 3.21% 1000 AGU/kg flour AMG 1100 0.52% 3.21%

FIGS. 4 to 6 show that the amyloglucosidases were used to produceglucose in different stages during the bread making process. FIG. 4shows that a RSD amyloglucosidase, such as AMG 1100, can be used toproduce sugar during dough mixing and dough proofing stages; whereas amore thermally-stable amyloglucosidase, such as Po-AMG, is much moreeffective in producing sugar during the actual baking stage (see FIGS. 5and 6). FIG. 6 showed that only the thermally-stable amyloglucosidase,such as Po-AMG used in this invention, produced a significant amount ofglucose during baking, whereas the conventional amyloglucosidase, suchas AMG 1100 from Aspergillus niger, was not able to produce significantamounts of glucose during baking since it was inactivated before thestarch granules were gelatinized. However, by using a combination of twodifferent types of amyloglucosidases, sugar, and most significantlyglucose, can be produced throughout the entire bread making process,which can maximize the sugar (particularly glucose) content in thefinished baked products, provide sufficient glucose for yeastfermentation during dough proofing, and give a desirable sweet taste ofthe finished baked products. Furthermore, as shown in the table above,significant amounts of maltose were produced, entirely during baking bythe thermally stable anti-staling maltogenic amylase. The high amount ofmaltose in the baked product also contributed to the flavor and taste ofthe baked products.

Example 3 Sugar Content and Its Effect on Anti-Staling Enzyme

A standard white pan bread formulation was prepared according to thefollowing formulation. Five different formulations were prepared byvarying the amount of sugar added to the formulation, the level ofanti-staling enzyme (NOVAMYL®), and the amount of amyloglucosidase,Po-AMG, and other ingredients were varied accordingly, as shown in theTables below.

White Staight Pan Bread Ingredients % of flour weight Gram Flour 100.0700 DEPENDOX ® AXC 0.06 0.42 Salt 2.0 14 Granulated sucrose Vary VaryCalcium Propionate 0.2 1.4 SSL (optional) 0.35 2.45 GMS-90 (optional)1.00 7 Soy Oil 2.0 14 1% BXP 25001 0.10 0.7 Yeast - dry Vary Vary WaterVary Vary Total Weight Vary Vary

Formula Variations Dough # 1 2 3 4 5 Po-AMG 0 AGU/kg flour 1000 AGU/kgflour Sucrose (% of 8% 4% 0% flour weight) Novamyl 0 2000 1000 2000 1000(MANU/kg flour) Yeast-dry (% of 3.0 3.0 2.5 2.5 2.0 flour weight) Water(% of 62.0 62.0 63.0 63.0 64.0 flour weight)

By using the inventive enzyme compositions, added-sugars can besignificantly reduced or completely removed from bread formulas, whichgreatly enhance the functionality of anti-staling enzymes, such asNOVAMYL®.

FIG. 7 shows that by reducing the amount of added sugar in the doughformula, the anti-staling function of NOVAMYL® is greatly enhanced. Inthis bake test, we showed that 1000 MANU/kg flour of NOVAMYL® at 4%added-sugar and 1000 AGU/kg flour of Po-AMG had similar crumb softeningeffect as 2000 MANU/kg flour of NOVAMYL with 8% added-sugar and 0 AGU/kgflour of Po-AMG; whereas 1000 MANU/kg flour of NOVAMYL® with 0%added-sugar and 1000 AGU/kg flour of Po-AMG performed significantlybetter than 2000 MANU/kg flour of NOVAMYL® with 8% added-sugar and 0AGU/kg flour of Po-AMG.

FIG. 8 shows the amounts of maltose produced by the anti-staling enzyme,NOVAMYL® in this baking test. Maltose is an end product of NOVAMYL®action, and the amount of maltose produced in the bread is directlyrelated to the functionality of the enzyme. The test results in FIG. 8show that by reducing or removing the added-sugar (i.e., the enzymeinhibitor) in the dough, more maltose was produced by NOVAMYL®, whichcorresponds with an increase in enzymatic activity and functionality.The increased activity of NOVAMYL® not only improved the anti-stalingeffect of the enzyme, but also resulted in high maltose levels in thebread, which made positive contributions to the taste and flavor of thefinished bread.

In this example, the inventive enzyme composition and without any addedsugar the level of yeast addition could be reduced from 3.0% to 2.0%,representing a 33% of yeast reduction.

Example 4 Combination of AMGs

This example examines the combination of a regular RSD amyloglucosidase,AMG 1100, and a thermally-stable amyloglucosidase, Po-AMG, in a 0%added-sugar baking A white bread dough was prepared using a no-timesystem. In this baking, 2000 MANU/kg flour of NOVAMYL® 3D, which is avariant of NOVAMYL®, was used as the anti-staling enzyme. AMG 1100 wasused as the RSD amyloglucosidase, along with the thermally-stableamyloglucosidase Po-AMG. The level of RSD amyloglucosidase, AMG 1100,was at 500 and 1000 AGU/kg flour, whereas the level of thethermally-stable amyloglucosidase, Po-AMG, was tested at 545 AGU/kg and1089 AGU/kg flour.

White Pan Bread - No-Time Ingredients % of flour weight Gram Flour 100.0700 DEPENDOX ® AXC 0.06 0.42 Salt 2.0 14 Granulated sucrose Vary VaryCalcium Propionate 0.3 2.1 SSL (optional) 0.35 2.45 GMS-90 1.00 7 SoyOil 2.0 14 Compressed Yeast Vary Vary Water Vary Vary Total Weight VaryVary

Formula Variations Dough # 1 2 3 4 Added Sucrose (% of 8 8 0 0 flourweight) UFP 250 (% of 0 0.25 0 0 flour weight) NOVAMYL ® 3D (% of 0 00.02 0.02 flour weight) Eversoft^(A) (% of 0 0 0.0025 0.0025 flourweight) AMG 1100 0 0 545 1089 (AGU/kg flour) Po-AMG (AGU/kg flour) 0 01000 500 Compressed Yeast (% of 5.75 5.75 4.0 4.0 flour weight) Water (%of 60.0 60.0 64.0 64.0 flour weight) ^(A)A bacterial amylase productfrom CorbionAfter baking, bread loaves were store in plastic bags for shelf lifestudy. For sugar analysis, bread crumbs were extracted with distilledwater, and the sugar content was analyzed on the Dionex HPLC system.FIG. 9 shows the sugar types and content in the bread. The resultsshowed that with the addition of the anti-staling enzyme and both typesof amyloglucosidases, AMG 1100 and Po-AMG, significant amounts ofglucose and maltose were produced in the bread. However, there was nodetectable amount of fructose in that bread made with 0% added sugar andthe invention enzyme compositions. FIG. 10 showed the calculatedsweetness based on the measured sugar contents for those bread samples.The results showed that with the addition of enzymes (both NOVAMYL® 3Dand the two AMGs), the bread with 0% of added-sugar were actuallysweeter than the control bread made with 8% added-sugar. FIGS. 11-12showed that with the addition of the enzymes, bread staling wassignificantly slowed, which can be measured by the decrease of crumbfirmness and increase of crumb resilience. Again, in this example, thedough made with the inventive enzyme formulation and 0% added sugarallowed a 30% yeast reduction, when compared to the dough with 8% addedsugar and without the inventive enzyme formulation.

Some of the improvements with respect to crumb resilience andadhesiveness (calculated from FIGS. 2, 3, 11, and 12) are summarized inthe following table:

AMG (AGU/kg flour) 0 500 1,000 2,000 5,000 FIG. 7 Resilience AMG 110028.2% 28.4% 28.8% 31.2% 32.3% 27.50% Po-AMG 28.2% 31.3% 33.3% 38.2%41.5% 35.20% Resilience  10%  16%  22%  28%   28% ImprovementAdhesiveness AMG 1100 12.4 12   11.9 10.4  8.5 Po-AMG 12.4 10.6  6.4 3.7  1.8 Adhesiveness  12%  46%  64%  79% Improvement

Example 5 Sensory Validation

In this Example, breads were prepared and evaluated for sensoryperception. A control bread made with 8% of added-sugar, in this casesucrose, and 6.65 Promu/kg Novamyl Pro, which is a variant of NOVAMYL®(available from Novozymes), was compared to a test bread made with 0% ofadded-sugar, 33.25 PROMU/kg Novamyl Pro, and a combination of a rawstarch degrading AMG (Gold Crust 3300 from Novozymes) and Po-AMG.

Sponge and Dough System % of flour weight 900 g dough Sponge Polar BearFlour 70 630.0 STARPLEX ® 0.25 2.3 SSL 0.375 3.4 Compressed Yeast 3 27.0Water 40.5 364.5 Total 114.13 1027.1 Dough Polar Bear Flour 30.00 270.0Sucrose Vary Vary Salt 2 18.0 Calcium Propionate 0.35 3.2 DEPENDOX ® AXC0.06 0.54 Soybean Oil 2 18.0 Compressed Yeast Vary Vary Water Vary Vary

Formula Variations on Dough Side Dough # 1 2 Added Sucrose (% 8 0 offlour weight) NOVAMYL ® Pro ^(A) 6.65 33.25 PROMU/kg flour Gold Crust3300 ^(B) 0 825 (AGU/kg flour) Po-AMG 0 756 (AGU/kg flour) YeastCompressed (% of 4.0 2.5 flour weight) Water (% of 14.0 18.0 flourweight) ^(A) A NOVAMYL variant from Novozymes ^(B) A Talaromycesemersoni amyloglucosidase from Novozymes

FIG. 13-16 showed a sensory comparison, with 46 panelists, of a controlbread made with 8% of granulated sucrose and 6.65 PROMU/kg of NOVAMYL®Pro, to a test bread made with 0% added-sugar, 33.25 PROMU/kg ofNOVAMYL® Pro, 825 AGU/kg of Gold Crust 3300, and 756 AGU/kg of Po-AMG.The results showed that the test bread with 0% added-sugar was scoredsignificantly higher in freshness, soft texture, and good taste. Asweetness evaluation also showed the test bread was tasted slightlysweeter than the control bread and it is more close to “just right”sweetness. Overall, about 90% of panelists (41 out of 46) preferred thetest bread made with zero percent of added-sugar (FIG. 15). The sugarcontents analysis (FIG. 16) again showed that the test bread made withno added sugar but with the inventive enzyme composition wasfructose-free.

1. A method of forming a bakery product, said method comprising:providing a dough comprising: yeast; an initial quantity of sugar; asource of starch; and a thermally-stable amyloglucosidase that exhibitsactivity at temperatures at which the starch gelatinizes; and an enzymeselected from the group consisting of: raw starch degradingamyloglucosidases; anti-staling amylases; and mixtures thereof; andbaking the dough for a time and temperature sufficient to yield thebakery product, said bakeryproduct having a final quantity of sugar thatis greater than said initial quantity of sugar.
 2. The method of claim1, wherein: said initial quantity of sugar is less than about 1.0% byweight, based upon the total weight of the dough taken as 100% byweight; said final quantity of sugar is at least about 5.0% by weight,based upon the total weight of the bakery product taken as 100% byweight; and said bakery product comprises less than about 0.5% by weightfructose, based upon the total weight of the bakery product taken as100% by weight.
 3. The method of claim 1, said thermally-stableamyloglucosidase being active at temperatures of from about 65° C. toabout 85° C.
 4. The method of claim 1, wherein said thermally-stableamyloglucosidase has a half-life (T_(1/2)) of from about 1 minute toabout 30 minutes at about 85° C.
 5. The method of claim 1, wherein saidthermally-stable amyloglucosidase is derived from strains selected fromthe group consisting of Penicillium oxalicum, Talaromyces emersonii,Talaromyces duponti, Talaromyces thermophilius, Clostridiumthermoamylolyticum, and Clostridium thermohydrosulfuricum.
 6. The methodof claim 1, wherein said enzyme is a raw starch degradingamyloglucosidase having a half-life (T_(1/2)) of from about 1 minute toabout 20 minutes at about 70° C.
 7. The method of claim
 1. wherein saidbakery product has a percent resilience of at least about 28% whenmeasured shelf life day
 7. 8. A dough useful for forming a yeast-raisedbakery product and comprising a source of starch, yeast, and water,wherein said dough comprises a thermally-stable amyloglucosidase thatexhibits activity at temperatures at which the starch gelatinizes; andan enzyme selected from the group consisting of raw starch degradingamyloglucosidases, anti-staling amylases and mixtures thereof.
 9. Thedough of claim 8, said thermally-stable amyloglucosidase being active attemperatures of from about 65° C. to about 85° C.
 10. The dough of claim8, wherein said thermally-stable amyloglucosidase has a half-life(T_(1/2)) of from about 1 minute to about 30 minutes at about 85° C. 11.The dough of claim 8, wherein said thermally-stable amyloglucosidase isderived from strains selected from the group consisting of Penicilliumoxalicum, Talaromyces emersonii, Talaromyces duponti, Talaromycesthermophilius, Clostridium thermoamylolyticum, and Clostridiumthermohydrosulfuricum.
 12. The dough of claim 8, wherein said enzyme isa raw starch degrading amyloglucosidase having a half-life (T_(1/2)) offrom about 1 minute to about 20 minutes at about 70° C.
 13. Ayeast-raised bakery product formed from flour, yeast, and water, whereinsaid product comprises: an inactivated, thermally-stableamyloglucosidase derived from a thermally-stable amyloglucosidase thatexhibits activity at temperatures at which starch gelatinizes; at leastabout 5% by weight sugar, based upon the total weight of the bakeryproduct taken as 100% by weight; and less than about 0.5% by weightfructose, based upon the total weight of the bakery product taken as100% by weight.
 14. The bakery product of claim 13, wherein saidinactivated thermally-stable amyloglucosidase is derived from athermally-stable amyloglucosidase having a half-life (T_(1/2)) of fromabout 1 minute to about 30 minutes at about 85° C.
 15. The bakeryproduct of claim 13, wherein said inactivated thermally-stableamyloglucosidase is derived from a thermally-stable amyloglucosidasethat is derived from strains selected from the group consisting ofPenicillium oxalicum, Talaromyces emersonii, Talaromyces duponti,Talaromyces thermophilius, Clostridium thermoamylolyticum, andClostridium thermohydrosulfuricum.
 16. The bakery product of claim 13,wherein said bakery product has a percent resilience of at least about28% when measured shelf life day
 7. 17. The bakery product of claim13,where said bakery product has a firmness of less than about 250 g offorce at shelf life day
 7. 18. The bakery product of claim 13, whereinsaid bakery product has an adhesiveness of from about 5 g*mm to about 25g*mm when measured at shelf life day 7.